Adenoviral and additional viralbased vectors

A number of additional viral types may also prove useful as vectors in the practice of gene therapy. Chief amongst these are the adenoviruses. Adeno-associated virus, the herpes virus, and a number of other viruses, are also being considered (Table 14.2).

Adenoviruses are relatively large, non-enveloped structures, housing double-stranded DNA as their genetic material. Their genome is much larger (approximately 35 kb) and more complex than those of retroviruses. In most instances, only a small fraction of this genome is removed when constructing an adenovirus-based vector. Upon cellular infection, adenoviral DNA becomes localized in the nucleus, but does not integrate into the host cell DNA. Usually, infection by wild-type adenoviruses is associated with, at worst, mild clinical symptoms in humans.

As potential vectors for gene therapy, adenoviruses display a number of both advantages and disadvantages (Table 14.3), and they have been used in over 300 gene therapy trials to date. Their major advantage relates to their ability to infect non-dividing cells efficiently and the usually

Table 14.3 Some characteristic advantages and disadvantages of adenoviruses as potential vectors for gene therapy. Refer to text for further details

Advantages

Adenoviruses are capable of gene transfer to non-

dividing cells They are easy to propagate in large quantities

High levels of gene expression are usually recorded

They are relatively stable viruses

Disadvantages

Adenoviruses are highly immunogenic in man

The duration of expression of transferred genes can vary, and is usually transient Infection of permissive cells with wild-type adenovirus usually results in cell lysis Adenoviruses display a broad selectivity in the cell types they can infect observed expression of large quantities of the desired gene products. However, the failure of the adenoviral-based DNA to integrate into the host cell generally means that its survival and, hence, the duration of gene expression, is limited. Adenovirus-based vectors, carrying various marker genes (i.e. a gene whose expression product is easily detected), have been administered to animals. Marker gene expression has been subsequently noted in various tissues, including heart, liver, muscle, bone marrow, central nervous system and endothelial cells. Duration of marker gene expression ranged from 2-3 weeks to several months.

Whereas short-term, high-level gene expression may be appropriate for some gene therapy applications, it would be of less use for the treatment of, for example, genetic diseases, where long-term gene expression would be required. This could be achieved, in theory, by repeat administration of the ad-enoviral vector. However, adenoviruses prompt a strong immune response, which limits the efficacy of repeat administration. Indeed, the gene therapy trial death in 1999, as mentioned previously, was apparently caused by a severe and unexpected inflammatory reaction to the adenoviral vector used.

Additional viruses that may prove of some use as future viral vectors include adeno-associated virus and herpes virus. Adeno-associated virus is a very small, single-stranded DNA virus: its genome consists of only two genes. It does not have the ability to replicate autonomously and can do so only in the presence of a co-infecting adenovirus (or other selected viruses).

Although it is found in the human population, it does not appear to be associated with any known diseases. Not surprisingly, only relatively small genes can be introduced into adeno-associated viral vector systems. Such systems, however, do provide a mechanism of gene transfer into non-dividing cells. It also seems to facilitate long-term expression of the transferred genetic material. In contrast to adenoviruses, nucleic acid transferred by adeno-associated viruses appears to be integrated into the recipient cell genome.

The herpes simplex virus represents another potential vector system that is receiving increased attention. Because herpes simplex virus is a neurotrophic virus, it may prove to be particularly useful in delivering genes to neurons of the peripheral and central nervous system. Upon infection, herpes simplex virus usually remains latent in non-dividing neurons, with its genome remaining in an unintegrated form. Thus far, it has proven difficult to generate a replication-incompetent, but yet viable, herpes simplex particle. Moreover, some of the replication-incompetent viruses generated still retain an ability to damage/destroy the cells they infect. Although herpes-based vector systems one day may prove useful in gene therapy, suitable and safe vector variants of herpes simplex virus must first be generated and tested.

An additional virus that has more recently gained some attention as a possible vector is that of the sindbis virus. A member of the alphavirus family, this ssRNA virus can infect a broad range of both insect and vertebrate cells. The mature virion particles consist of the RNA genome com-plexed with a capsid protein C. This, in turn, is enveloped by a lipid bilayer in which two additional viral proteins (E1 and E2) are embedded. The E2 polypeptide appears to mediate viral binding to the surface receptors of susceptible cells. The major mammalian cell surface receptor it targets appears to be the highly conserved, widely distributed laminin receptor.

The sindbis virus is simple, robust, capable of infecting non-dividing cells and generally supports high levels of gene expression. However, it does display a broad host range and, hence, lacks the inherent targeting specificity characteristic of an idealized viral vector.

Recently, a novel recombinant sindbis virus, displaying altered host cell specificity, has been generated. Scientists inserted a nucleotide sequence coding for the IgG binding domain of Sta-phyloccus aureus into the E2 viral gene. Disruption of the E2 gene renders its protein product incapable of binding laminin (hence, destroying the natural viral tropism). However, the protein A domain allows the chimaeric E2 product to bind monoclonal antibodies. This altered virus may prove to be a useful generic or 'null' vector, potentially capable of being specifically targeted to any desired cell type. This would simply necessitate pre-incubation of the virus with monoclonal antibodies raised against a surface antigen unique to the proposed target cell population (Figure 14.6).

Figure 14.6 Generation of engineered sindbis virus capable of being targeted to bind specific cell types. (a) A simplified depiction of the virus, displaying the surface E2 protein. (b) Genetic engineering facilitates disruption of the E2 gene by incorporation of the IgG binding domain of protein A. (c) Incubation of such engineered viral particles with most monoclonal antibody types results in effective immobilization of the antibody on the viral surface. Thus, the engineered viral vector should be targetable to any specific cell type simply by its preincubation with monoclonal antibodies that selectively bind a surface antigen uniquely associated with the target cell

Modified E 2 protein, now containing the IgG binding domain of Protein A

Modified E 2 protein, now containing the IgG binding domain of Protein A

Figure 14.6 Generation of engineered sindbis virus capable of being targeted to bind specific cell types. (a) A simplified depiction of the virus, displaying the surface E2 protein. (b) Genetic engineering facilitates disruption of the E2 gene by incorporation of the IgG binding domain of protein A. (c) Incubation of such engineered viral particles with most monoclonal antibody types results in effective immobilization of the antibody on the viral surface. Thus, the engineered viral vector should be targetable to any specific cell type simply by its preincubation with monoclonal antibodies that selectively bind a surface antigen uniquely associated with the target cell

Binding of the monoclonal antibody to the protein A domain would ensue and the immobilized monoclonal antibody would dictate the cell type targeted.

Initial studies using this system have proved encouraging. The altered virus (without associated monoclonal antibody) failed to infect a wide variety of human cell lines. By initially incubating with monoclonal antibody of the appropriate specificity, however, the viral particles were capable of efficiently transducing cells expressing surface receptors such as CD4, CD33 and human leukocyte antigen.

A number of other issues must now be addressed including determining if the IgG-protein A affinity is sufficiently high to keep the antibody associated with the virus in vivo. The full potential of this approach will also require more detailed characterization of surface markers uniquely associated with different cell types. However, the approach exemplifies the types of technical innovation now being introduced that will make second-generation vectors more suited to their role in gene therapy.

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